Implication of HMGR in homeostasis of sequestered and de novo produced precursors of the iridoid biosynthesis in leaf beetle larvae

Insects employ iridoids to deter predatory attacks. Larvae of some Chrysomelina species are capable to produce those cyclopentanoid monoterpenes de novo. The iridoid biosynthesis proceeds via the mevalonate pathway to geranyl diphospate (GDP) subsequently converted into 8-hydroxygeraniol-8-O-beta-D-glucoside followed by the transformation into the defensive compounds. We tested whether the glucoside, its aglycon or geraniol has an impact on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzyme of the mevalonate pathway and also the iridoid biosynthesis. To address the inhibition site of the enzyme, initially a complete cDNA encoding full length HMGR was cloned from Phaedon cochleariae. Its catalytic portion was then heterologously expressed in Escherichia coli. Purification and characterization of the recombinant protein revealed attenuated activity in enzyme assays by 8-hydroxygeraniol whereas no effect has been observed by addition of the glucoside or geraniol. Thus, the catalytic domain is the target for the inhibitor. Homology modeling of the catalytic domain and docking experiments demonstrated binding of 8-hydroxygeraniol to the active site and indicated a competitive inhibition mechanism. Iridoid producing larvae are potentially able to sequester glucosidically bound 8-hydroxygeraniol whose cleavage of the sugar moiety results in 8-hydroxygeraniol. Therefore, HMGR may represent a regulator in maintenance of homeostasis between de novo produced and sequestered intermediates of iridoid metabolism. Furthermore, we demonstrated that HMGR activity is not only diminished in iridoid producers but most likely prevalent within the Chrysomelina subtribe and also within the insecta.     

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.     

De novo biosynthesis versus sequestration: a network of transport systems supports in iridoid producing leaf beetle larvae both modes of defense

In the larval chrysomelines the de novo synthesis of monoterpenoids (iridoids) is believed to represent the ancestral state in the evolution of chemical defenses. Here we demonstrate that the iridoid producing larvae of Plagiodera versicolora and Phratora laticollis have the potential to sequester precursors from food. In nature, iridoids may even have a dual origin, namely plant-derived and de novo produced. The ability to sequester plant-derived precursors was proved by (i) (13)C-labelling of the terpenoids in the food plant, (ii) by larval feeding on leaves impregnated with analogs and labelled putative precursors for iridoid biosynthesis; and (iii) by injection of the precursors into the hemolymph followed by mass spectroscopic analysis of their distribution in the hemolymph, defensive secretion, and faeces. The experimental findings support a network of transport systems which allows a broader range of glucosides to enter and to leave the hemocoel, while only the appropriate precursor, 8-hydroxygeraniol-8-O-beta-d-glucoside, is channelled to the reservoir and processed to iridoids. The dual system of de novo biosynthesis and sequestration of phytogenic precursors may have favoured the larvae to shift from one host plant to another without losing their defense.     

Salicyl alcohol oxidase of the chemical defense secretion of two chrysomelid leaf beetles. Molecular and functional characterization of two new members of the glucose-methanol-choline oxidoreductase gene family

Salicyl alcohol oxidase is an extracellular enzyme that occurs in glandular reservoirs of chrysomelid leaf beetle larvae and catalyzes the formation of salicylaldehyde, a volatile deterrent used by the larvae against predators. Salicyl alcohol is the hydrolysis product of salicin, a plant-derived precursor taken up by the beetle larvae from the leaves of willow and poplar trees. The cDNA encoding salicyl alcohol oxidase from two related species Chrysomela tremulae and Chrysomela populi has been identified, cloned, and expressed in an active form in Escherichia coli. The open reading frame of 623 amino acids begins in both enzymes with an N-terminal signal peptide of 21 amino acids. Sequence comparison has revealed that salicyl alcohol oxidase belongs to the family of glucose-methanol-choline oxidoreductase-like sequences with mostly unknown function. Enzymes of this family share similar overall structure with an essentially identical FAD-binding site but possess different catalytic activities. The data suggest that salicyl alcohol oxidase, essential for the activation of the plant-derived precursor salicin, was originally recruited from an oxidase involved in the autogenous biosynthesis of iridoid monoterpenes and found in related chrysomelid leaf beetle species.     

To be or not to be convergent in salicin-based defence in chrysomeline leaf beetle larvae: evidence from Phratora vitellinae salicyl alcohol oxidase

Glandular chemical defence relying on the action of salicylaldehyde is characteristic for Chrysomela leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in Chrysomela species. We here identified and characterized SAO of Phratora vitellinae, the only species outside the genus Chrysomela that produce salicylaldehyde as a defensive compound. Although Chrysomela and Phratora are not closest relatives, their SAOs share glucose-methanol-choline oxidoreductase (GMC) affiliation, a specific GMCi subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both Chrysomela and Phratora. Closely related species of Chrysomela and P. vitellinae use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.     

Characterization of an extracellular salicyl alcohol oxidase from larval defensive secretions of Chrysomela populi and Phratora vitellinae (Chrysomelina)

Larvae of a number of chrysomelid leaf beetles sequester phenol glucosides such as salicin from their food plants, i.e. Salix and Populus spp. Salicin is hydrolyzed in the glandular reservoir of the defensive glands. The resulting salicyl alcohol (saligenin) is oxidized by an extracellular oxidase. The product salicylaldehyde accumulates as major defensive compound. The secretions from Chrysomela populi and Phratora vitellinae were preserved in saturated ammonium sulfate solution and subjected to micro-purification of the oxidase by means of electrophoretic methods. The enzyme from P. vitellinae has a native M(r) of 334,000 and a subunit M(r) of 79,000 indicating a tetrameric enzyme. The isoelectric points of the enzymes from C. populi and P. vitellinae are at pH 5.4 and 5.2, respectively. In the oxidation of salicyl alcohol oxygen functions as electron acceptor yielding hydrogen peroxide as product. Hydrogen peroxide does not accumulate in native secretions but appears to be degraded most likely by a catalase. The oxidases from the two species show broad pH optima in the range 5.5 to 6.5, they oxidize salicyl alcohol as main substrate. Minor substrates are several ortho-substituted and to a lesser extent meta- but not para-substituted benzyl alcohols. In the presence of 8-hydroxygeraniol only trace amounts of the respective aldehyde are formed. The Km values of salicyl alcohol are 132 mM (C. populi) and 63 mM (P. vitellinae). The extracellular enzyme, which is functionally related to fungal aryl alcohol oxidase (EC 1.1.3.7) and vanillyl alcohol oxidase (EC 1.1.3.38) was named salicyl alcohol oxidase. The continuous formation of salicylaldehyde in the glandular reservoir can be compared to the operation of an enzyme reactor. Due to its low aqueous solubility the produced aldehyde steadily leaves the aqueous reaction fluid and builds up an organic phase which may account for 15% of the total liquid volume of the secretion.     

Always being well prepared for defense: The production of deterrents by juvenile Chrysomelina beetles (Chrysomelidae)

In response to herbivores, plants produce a variety of natural compounds. Many beetle species have developed ingenious strategies to cope with these substances, including colonizing habitats not attractive for other organisms. Leaf beetle larvae of the subtribe Chrysomelina, for example, sequester plant-derived compounds and use them for their own defense against predators. Using systematically modified structural mimics of plant-derived glucosides, we demonstrated that all tested Chrysomelina larvae channel compounds from the gut lumen into the defensive glands, where they serve as intermediates in the synthesis of deterrents. Detailed studies of the sequestration process revealed a functional network of transport processes guiding phytochemicals through the larval body. The initial uptake by the larvae’s intestine seems to be fairly unspecific, which contrasts sharply with the specific import of precursors into the defensive glands. The Malpighian tubules and hind-gut organs facilitate the rapid clearing of body fluid from excess or unusable compounds. The network exists in both sequestering species and species producing deterrents de novo. Transport proteins are also required for de novo synthesis to channel intermediates from the fat body to the defensive glands for further conversion. Thus, all the tools needed to exploit host plants’ chemistry by more derived Chrysomelina species are already developed by iridoid–de novo producers. Early intermediates from the iridoid–de novo synthesis which also can be sequestered are able to regulate the enzyme activity in the iridoid metabolism.     

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.     

Iridoid patterns in Galium L. and some phylogenetic considerations

From 19 species of Galium, members of 6 European sections of the genus, 24 compounds were isolated, namely 16 iridoid glucosides, 2 secoiridoid glucosides and 6 triterpene saponins (the later found only in G. rivale (Sibth. & Sm. Griseb.) The iridoid content was analyzed by thin layer chromatography - densitometry. An effort was made to clarify interspecies relationships, based on the obtained results and previous data. Generally, a nearly uniform iridoid pattern in the studied species was observed. Nevertheless, some distinctions gave reason the following chemical characters to be treated as taxonomic markers: iridoids, secogalioside (characteristic of G. mollugo group), iridoids V1 and V2 (G. humifusum Bieb. and G. verum L.), 6-acetylscandoside (G. incurvum group and G. verum) and the triterpene saponins, rivalioside A and rivalioside C (characteristic of G. rivale). The studied species regarding to the iridoids could be attributed to three lines of evolutionary differentiation. One line is leading to the differentiation of G. rivale. It contains specific triterpenoids as well as iridoid acids, which show parallel development of both glyceraldehyde 3-phosphate/pyruvate and mevalonate biosynthetic routes in this species. A second line includes G. mollugo and G. incurvum species groups and the species G. humifusum and G. verum. Variety of iridoid esters, hydroxy and carboxy derivatives of iridoids and secoiridoids characterised this line. Third line comprises the remaining studied species, members of different sections and species groups. They posses a nearly identical iridoid pattern, which suggests a convergent evolution regarding to the iridoids.     

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant–insect interactions.     

Food utilization values of gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) larvae infected with the microsporidium Vairimorpha sp. (Microsporidia: Burenellidae)

Infection of the gypsy moth, Lymantria dispar, with the microsporidium Vairimorpha sp. strongly influences the development of the host in ways typical of many species of terrestrial entomopathogenic Microsporidia; growth is reduced while development time is extended in infected insects. The appearance of the different stages of the parasite in the host relative to the elapsed time after oral infection, as well as the influence of the parasite proliferation on food utilization of the host, were examined. At 3 days postinfection, midgut muscle cells were infected with primary spores, and the fat body tissues contained meronts, sporonts, and primary spores. Many more fat body cells contained vegetative stages and primary spores at 4 and 5 days postinfection, and diplokaryotic spores and immature octospores were also present. Approximate digestibility of infected larvae increased during this time period, whereas the conversion of ingested and digested food to body substance decreased. The relative growth rate of infected and uninfected groups did not differ significantly between 4 and 5 days postinfection, although the relative consumption rate in infected L. dispar larvae was higher. Between 8 and 10 days postinfection, the relative growth rate of uninfected larvae increased. The infected group did not demonstrate this increase at a time period characterized by maturation of diplokaryotic spores and octospores in larval fat body tissues. Total body weight of uninfected larvae remained higher than that of infected larvae after 8 days postinfection.     

Influence of larvae of Gastrophysa viridula on the distribution of conspecific adults in the field

1. Previous laboratory bioassays indicate that exocrine glandular secretions of larvae of Gastrophysa viridula repel conspecific adults and deter them from feeding and oviposition. The present study was conducted to investigate the influence of larvae of G. viridula on conspecific adults in the field.
2. Within the G. viridula population studied, two generations were observed in a year. Occurrence of the different developmental stages overlapped temporally.
3. Some individual plants of Rumex obtusifolius , the host of the G. viridula population studied, grew so close to each other that they were considered as a plant group. When investigating the spatial distribution of larvae and adults within such plant groups, larvae were rarely found on plant groups on which adults were feeding.
4. A field experiment revealed that adults avoided plants of R. obtusifolius infested by conspecific larvae of the second and third instar. Adults still avoided these damaged plants when larvae had left them for pupation.
5. Gastrophysa viridula avoided oviposition on leaves and plants infested by conspecific larvae. Larvae of the second instar significantly deterred oviposition when present at a high density (33.3 larvae/dm²), whereas larvae of the first instar did not deter oviposition of conspecifics at either density tested. The oviposition deterring effect was also observed when just exocrine glandular secretion of larvae of the second instar was applied to the leaves in amounts equivalent to a density of 33.3 larvae/dm².
6. Availability of food ( R. obtusifolius ) largely exceeded its exploitation in each group of plants examined. This may be due to either the spatial separation of adults and larvae or the low population density observed on these plant groups.     

Structural studies on 10-hydroxygeraniol dehydrogenase: A novel linear substrate-specific dehydrogenase from Catharanthus roseus

Conversion of 10-hydroxygeraniol to 10-oxogeranial is a crucial step in iridoid biosynthesis. This reaction is catalyzed by a zinc-dependent alcohol dehydrogenase, 10-hydroxygeraniol dehydrogenase, belonging to the family of medium-chain dehydrogenase/reductase (MDR). Here, we report the crystal structures of a novel 10-hydroxygeraniol dehydrogenase from Catharanthus roseus in its apo and nicotinamide adenine dinucleotide phosphate (NADP⁺) bound forms. Structural analysis and docking studies reveal how subtle conformational differences of loops L1, L2, L3, and helix α9' at the orifice of the catalytic site confer differential activity of the enzyme toward various substrates, by modulating the binding pocket shape and volume. The present study, first of its kind, provides insights into the structural basis of substrate specificity of MDRs specific to linear substrates. Furthermore, comparison of apo and NADP⁺ bound structures suggests that the enzyme adopts open and closed states to facilitate cofactor binding.     

A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle

Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-SUGAR GLYCOSYLTRANSFERASE6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.     

Iridoid glycosides from Penstemon richardsonii

Five ester iridoids of the valeriana type have been isolated from dried leaves of Penstemon richardsonii. The structures have been elucidated by FAB MS ¹H NMR and ¹³C NMR spectroscopy and by enzymatic cleavage of the glycosidic linkages. Besides the known iridoids penstemide penstemide-aglucone 8-epi-valerosidatum and serrulatoside a new iridoid penstebioside (penst of an iridoid aglycone attached to cellobiose.     

Selective sequestration of iridoid glycosides from their host plants in Longitarsus flea beetles

We investigated in eight species of the flea beetles genus Longitarsus (Coleoptera, Chrysomelidae) whether the beetles take up iridoid glycosides from their host plants of the Lamiaceae, Plantaginaceae, and Scrophulariaceae. Five of the beetle species, L. australis, L. lewisii, L. melanocephalus, L. nigrofasciatus, and L. tabidus, could be shown to sequester iridoid glycosides in concentrations between 0.40 and 1.55% of their dry weight. Eight different iridoid glycosides, acetylharpagide, ajugol, aucubin, catalpol, 8-epi-loganic acid, gardoside, geniposidic acid, and harpagide could be identified in the host plants, yet only aucubin and catalpol are sequestered by the beetles. No iridoid glycosides could be detected in the beetles if neither aucubin nor catalpol were present in the host plant, as in L. minusculus on Stachys recta (acetylharpagide only) and in L. salviae on Salvia pratensis (no iridoid glycosides). In one beetle species, L. luridus, we could not detect any iridoid glycosides although its field host, Plantago lanceolata, had considerable amounts of aucubin and catalpol plus two further iridoids. The five sequestering Longitarsus species differ in their capacity to store the compounds and in their affinity for catalpol relative to aucubin.     

Bioinformatics study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase (HMGR) gene in Gramineae

Isoprenoids or terpenoids are synthesized by two important units' including dimethylallyl diphosphate and isopentenyl diphosphate (IPP). Plants use two different methods for formation of IPP, which is a cytosolic and a plastidial method. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyzes the conversion of HMG-CoA to mevalonate, which is the first stage in the cytosolic pathway for biosynthesis of isoprenoid in plants. In this study, a total of fifty HMGR protein sequences from Gramineae and three animal samples including human, mouse and fruit fly were aligned and analyzed by computational tools to predict the protein properties, such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. Sequence comparison analysis revealed that there is high identity between plants and animals. Three catalytic regions including L domain, N domain and S domain were detected by structural modeling of HMGR. The tertiary structure model of Oryza sativa HMGR (Accession Number: NP_001063541) was further checked by PROCHECK algorithm, and showed that 90.3?% of the amino acid residues were located in the most favored regions in Ramachandran plot, indicating that the simulated three-dimensional structure was reliable. Phylogenetic analysis indicated that there is a relationship among species of Gramineae and other organisms. According to these results, HMGRs should be derived from a common ancestor.     

Chemical Traits of Hemiparasitism in Odontites luteus

The study of the monoterpene glycosides content of Odontites luteus has shown the presence of a total of fifteen iridoid glucosides. The presence of compounds 1  –  5 and 7  –  10 is perfectly on‐line with both the biogenetic pathway for iridoids biosynthesis in Lamiales and the current botanical classification of the species. On the other side, the presence of compounds like agnuside ( 6 ), adoxosidic acid ( 11 ), monotropein ( 12 ), 6,7‐dihydromonotropein ( 13 ), methyl oleoside ( 14 ) and methyl glucooleoside ( 15 ) is of high interest because, first of all, they have never been reported before in Lamiales. In second instance, the majority of the last compounds are formally derived from a different biogenetic pathway which involves deoxyloganic acid/loganin and led to the formation of decarboxylated iridoid showing the 8β‐configuration. Furthermore, a second abnormality was found during our study and this regards compounds 14 and 15 which are seco‐iriodids and thus not typical for this family. The presence of these unusual compounds, biogenetically not related to species belonging to Lamiales, is a clear evidence of the metabolites transfer from the hosts. In fact, the collection area is also populated by species belonging to Oleaceae and Ericaceae which could be the possible hosts since the biosynthesis of seco‐iridoids and or iridoids related to deoxyloganic acid/loganin pathway, with the 8β‐configuration, is well documented in these species.     

Iridoid patterns of genus Plantago L. and their systematic significance

The distribution of 14 iridoid glucosides in 14 Plantago L. species (44 samples corresponding to 18 taxa) was shown. P. tenuiflora and P. gentianoides were studied for iridoids for the first time. The iridoid patterns showed a good correlation with morphological and other chemical features of the representatives of genus Plantago. The studied species are grouped together according to the iridoid patterns: species containing mainly aucubin (P. major, P. cornuti, P. gentianoides); species containing aucubin and aucubin derivatives (P. subulata, P. media); species containing aucubin and catalpol (P. lanceolata, P. altissima, P. argentea, P. lagopus, P. atrata); species containing aucubin and plantarenaloside (P. afra, P. scabra).